Pharmaceuticals and assays using enzyme subunits

ABSTRACT

A method of releasing an agent for example, a chemotherapeutic, under predetermined conditions by protecting the agent within a lipid structure such as a liposome, causing lipase activity to be constituted by combining two or more components, e.g., recombinant N- or C-terminal  Clostridium perfringens  alpha-toxin fragments, one of these components being conjugated to a targeting molecule e.g., an antibody which binds to a target such as a tumor antigen. The lipid structure is then exposed to the constituted lipase activity such as to release the agent. Also disclose are materials and kits for use in the method.

This application is a 33 U.S.C. 371 of PCT/GB96/00380 filed Feb. 21,1996, and claims benefit to GB 95 03486.4, filed Feb. 22, 1995.

The present invention relates to methods for releasing an agent underpre-determined conditions, for example at a pre-determined site or inthe presence of a pre-determined material, and in particular forreleasing an agent for therapeutic, diagnostic or investigativepurposes. The invention further relates to pharmaceutical compositionsincorporating such methods, materials and kits for use in such methods.It is frequently desirable in the bioscience field to be able to depositor release a particular agent under, pre-determined conditions, forinstance at a specific site within an organism or to mark the presenceor absence of an analyte during an assay. At present such specificity isfrequently achieved by use of antibodies bound directly to activeagents. For instance tumour associated monoclonal antibodies (MABS) havebeen used to selectively carry chemotherapeutic drugs to tumour cells.Clinical studies have investigated the delivery of methotrexate inpatients colorectal carcinoma (Ballantyne et al. 1988. Int. J. Cancer,42: 103-108) and also the use of adriamycin (see “Principles of CancerBiotherapy” Ed. Oldham, R. K., Pub. Raven Press, New York, 1987).Similarly. MABS conjugated to toxins such as ricin, abrin, Pseudomonastoxin, Diptheria toxin and other have also been used as anti-canceragents. Studies in vitro and in vivo have indicated that such conjugatescan be extremely toxic to tumour cells ( Roffler et al. 1991. CancerRes. 51:4001-4007; Embleton et al 1991; Bri. J. Cancer 63:670-674).

The use of MABS to provide selectivity avoids the side-effect problemsassociated with traditional chemotherapeutic treatment of cancer eitherin metastatic disease or in an adjuvant or primary setting. However, amajor problem arises because many agents require internalisation beforekilling the target cell. Additionally immunotoxins usually give rise tounacceptable toxicity due to interaction with non-target cells duringpassage to the site.

A potential alternative delivery system for selected agents is basedaround the use of synthetic liposomes. Liposomes were originallydescribed in 1974 (Bangham e al. Methods Membr. Biol. 1: 1-68).Liposomes consist of one or more phospholipid bilayers arranged inconcentric rings of alternate aqueous spaces. Many compounds (both lipidand Water soluble) including cancer chemotherapeutics, antimicrobialdrugs, enzymes, hormones and nucleic acids have been incorporated intoeither the aqueous or lipid phase of liposomes. The behaviour ofdrug-containing liposomes in animal and human subjects has formed thesubject of several studies (Gregoriadis 1990, Immunol. Today 11: 89-97).

Thus liposomes offer considerable promise as vehicles for delivery ofagents for use in a variety of applications including biochemical andimmunological assays, diagnosis, and also pharmaceutical deliverysystems for eternal and parenteral use. Unfortunately their applicationis undermined by the difficulty associated with selectively releasingtheir contents at a specific time or location.

The present invention has now provided methods for releasing a selectedagent at a specific disease site or at a specific time or location andpharmaceutical compositions incorporating such methods, kits andmaterials for use in such methods, which seek to address some, and inpreferred forms all, of the aforementioned problems.

According to a first aspect of the present invention there is provided amethod of releasing an agent under predetermined conditions comprisingthe steps of protecting the agent within a lipid structure, causinglipase activity to be constituted in response to the predeterminedconditions, and exposing the lipid structure to the constituted lipaseactivity such as to release the agent.

By lipase is meant any enzyme which hydrolyses lipids and includes, butis not limited to. enzymes which hydrolyse complex lipids such asphospholipids and grycolipids.

The term constituted as used herein is intended to denote localised,created or significantly increased i.e. a significant achievement orincrease in lipase activity is initiated when the predeterminedconditions are met.

A large number of naturally occurring lipases are known. For instancemany gram-positive and negative bacteria produce enzymes havingphospholipase C (PLC) activity. These enzymes hydrolyse phospholipidswith varying efficiencies and posses a variety of haemolytic and lethalproperties which generally makes them unsuitable for administration toliving subjects.

One characterised enzyme is Clostridium perfringens alpha-toxin (CPAT).CPAT promotes direct lysis of certain mammalian cells and is the mosttoxic PLC described to date (see McDonel, J.L. (1986) pp 617-655“Pharmacology of Bacterial Toxins” Eds. Dorner & Drews, Pub. PergamonPress, Oxford). CPAT is a peptide containing 370 amino acids.

Preferably the lipid structure employed by the present inventioncomprises a phospholipid membrane defining a core. More preferably thelipid structure is a liposome. The agent to be released is chosen inaccordance with the precise application in which the invention is beingemployed, however the nature of the agent must be such that it isprotectable by a lipid structure.

Preferably the lipase activity employed in the present inventioncomprises a PLC activity, and more preferably is derived from CPAT. CPATactivity has not previously been demonstrated against liposomes; howeverthe inventors of the present invention have shown that CPAT hassignificant activity against liposomes.

Preferabiy the lipase activity employed in the present invention isconstituted by combining two or more components whereby the lipaseactivity of the product formed by the components is greater than the sumof the individual components, or alternatively the lipase activity isconstituted at a specific location normally with much less or no lipaseactivity by virtue of the localisation of the lipase either as aholoenzyme or a combination of two or more components in either case incombination with a targeting molecule.

More preferably the components correspond to, or are derived from, anactive lipase holoenzyme such that their recombination recovers all orpart of the activity of the holoenzyme lipase preferably at a specificlocation. These components may both be proteins—however the inventionembraces all systems wherein lipase activity is enhanced. localised orrecovered by the combination of two or more components, includingnon-protein components such as co-factors.

Most preferably the components are derived from or include CPAT.

The N-terminal two-thirds of CPAT shares sequence homology with thephosphatidylcholine-PLC from Bacillus cereus. It has been demonstratedthat N-terminal recombinant truncated CPAT (aa 1-249) retainsphosphatidylcholine hydrolysing activity but has reducedsphingomyelinase activity and is neither haemolytic nor lethal. (Titballet al 1991. Infect. Immun. 59:1872-1874). Recombinant protein comprisingthe C-terminal third of CPAT (aa 247-370) is devoid of sphingomyelinaseand haemolytic activity and is not toxic for murine lymphocytes (Titballet al. 1993, FEMS Microbiology Letters 110: 4550). It has beendemonstrated that haemolytic activity (as assessed by an in vitro murineerythrocyte lysis assay) can be restored when the N-terminal andC-terminal recombinant proteins are added together (reconstituted CPAT).

The inventors of the present invention have shown that reconstitutedCPAT has significant activity against liposomes.

The pre-determined conditions of the present invention may require thatthe agent be released only in the vicinity of a tumour or pathogen, orin the presence of an anaiyte or DNA sequence, or under any othersuitable detectable condition.

Preferably the achievement of predetermined conditions is causallyrelated to the constitution of lipase activity at a specific location byconjugating at least one of the lipase components or the holoenzyme to atargeting molecule capable of specific binding to a predetermined targetunder the pre-determined conditions. Suitable targeting moleculesinclude antibodies, antigens, receptors, ligands and nucleic acid probesor primers.

Thus the achievement of conditions may be conveniently related (via aspecific antigen-antibody binding event, or the annealing of anucleotide probe to a specific sequence, or to some other specificphysical process) to the constitution of lipase activity in the presenceof a predetermined target. Thereafter the addition of suitable lipidstructures e.g. liposomes, to the system will effectively lead totarget-induced liposomal lysis.

Thus in one embodiment of the invention, which may be used to release anagent at a predetermined target site, the targeting molecule is anantibody which has been raised such as to bind to an antigen on a targetsite. The antibody is conjugated to a first lipase component such thatthe component binds at the site. A second lipase component is also addedto the system and binds at the site. A second lipase component is alsoadded to the system and binds to the first such that lipase activity isconstituted at the site without having had fully active lipasecirculating in the system. Liposome containing a suitable agent may beadded to the system such that they are lysed on contacting theconstituted lipase thereby releasing the agent locally at the site.

It should be noted that the second lipase component may be addedindependently of in association with, or as an integral part of theliposomes. In another embodiment of the invention which may be used torelease an agent at a predetermined target site, the targeting moleculeis an antibody which has been raised such as to bind to an antigen on atarget site. The antibody is conjugated to a lipase holoenzyme such thatthe holoenzyme binds at the site. Liposomes containing a suitable agentmay be added to the system such that they are lysed on contacting theconstituted lipase thereby releasing the agent locally at the site.

Thus these embodiments have in vivo applications for the treatment orlocating of a disease through the targeted delivery of membrane-lyticenzyme activity. Unbound anti-body-component conjugate may be clearedfrom the system prior to adding the second component so as to ensureonly local constitution of lipase activity. Specific ablation of thediseased cells or organism may then be achieved by addition of liposomescontaining disease modulating compounds. Use of the present inventionagainst tumours may therefore provide an improved killing index due tothe ability to release a high local concentration of compound at thetumour and also possible beneficial by-stander effects whereinnon-antigen bearing cancer cells in the immediate locality of the tumourwill also be the subject of chemotherapeutic killing.

Suitable compounds may include reporter molecules, cytotoxic drugs,lymphokines. anti-inflammatories, anti-fungals, anti-malarials and otherdrugs combating infectious diseases. synthetic oligonucleotides, nucleicacids (e.g. plasmids) etc, additional antibodies active as immunotoxins,enzymes for conversion of inactive to active pharmaceutical compoundsetc. The precise compounds to be used will occur to those skilled in theart according to the problem to be solved.

In a second embodiment of the invention, which may be used for detectingthe presence of an antigen in a system, the targeting molecule is anantigen conjugated to a first lipase component. The antigen-1stcomponent conjugate is mixed with antibody raised against authenticantigen such that the antibody binds to the conjugated antigen. Thepresence of the bound antibody sterically prevents the constitution oflipase activity in the presence of a second lipase component. When theantibody/antigen-component complex is in the presence of authenticantigen the antibody is sequestered by and binds to the authenticantigen. This means that lipase activity will be constituted in thepresence of the second lipase component. This event (and hence thepresence of authentic antigen) can be detected by addition of liposomescontaining a suitable marker e.g. dye, to the system.

Thus this embodiment has in vitro application as an homogenous assaysystem for the detection of biological or other analytes. The antigenmay be conjugated to one of the lipase constituents by recombinant orbiochemical techniques.

In another embodiment of the invention, which may be used for detectingthe presence of an antigen in a system, the targeting molecule is anantibody conjugated either to a first lipase component or a holoenzyme.The antibody-lipase conjugate may be attached to a specific antigenwhich has become associated with a solid phase either by direct bindingto the solid phase or by binding via an antibody or other intermediatemolecule. The presence of antigen can be measured following attachmentof the conjugate and washing-off or elution of the excess conjugate bythe addition of liposomes containing a suitable marker eg dye.

As an alternative for the detection of an antigen in a system, theantigen could also be attached to a solid phase either directly byabsorption or chemical linkage or indirectly via another antibody orbinding agent which is, in turn, attached to a solid phase. Theantibody-lipase conjugate comprising either the complete lipase or onecomponent is then added followed by if required, the second lipasecomponent and followed by suitable compound-containing liposomes.

In a third embodiment, which may be used for detecting the presence orlocation of a specific nucleotide sequence in a system, a suitablecomplementary probe is attached to one or both lipase components suchthat annealing of the probe or probes to the sequence causes lipaseactivity to be constituted at that site. This event (and hence thepresence of the sequence) can be detected by addition of liposomescontaining a suitable marker e.g. dye to the system.

As an alternative for the detection of a specific nucleotide sequence ina system the nucleotide sequence could also be attached to a solid phaseeither directly by absorption or chemical linkage or indirectly via acomplementary nucleotide sequence or an antibody or binding agent whichis, in turn, attached to a solid phase. The probe-lipase conjugatecomprising either the complete lipase or one component is then addedfollowed by if required, the second lipase component and followed bysuitable compound-containing liposomes.

Also embraced by the present invention are materials for use in themethods above.

Thus in a second aspect of the invention there is provided a firstlipase component capable of combining with a second lipase componentsuch that the lipase activity of the product formed by the components isgreater than the sum of the individual components, said first lipasecomponent being conjugated to targeting molecule capable of specificbinding to a predetermined target.

Also embraced by the present invention are pharmaceutical preparationscomprising a targeting molecule conjugated with a lipase holoenzyme orlipase component and liposomes containing pharmaceutically activecompounds or compounds capable of conversion into pharmaceuticallyactive molecules.

Also embraced by the present invention are kits for use in the methodsabove.

Thus in a third aspect of the invention there is provided a kit for usein the methods above comprising a first lipase component capable ofcombining with a second lipase component such that the lipase activityof the product formed by the components is greater than the sum of theindividual components, said first lipase component being conjugated totargeting molecule capable of specific binding to a predeterminedtarget, and further comprising the second lipase component.

Preferably the kit still further comprises liposomes containing asuitable agent for use in the methods above.

Preferably the lipase components are CPAT holoenzyme or N-terminalrecombinant CPAT and C-terminal recombinant CPAT as herein beforedescribed.

A range of alternative lipases may be applicable for use in the currentinvention. These alternatives include lipases from bovine and porcinepancreas, bee venom, Crotalus venom and include phospholipase B fromS.violaceoruber and phospholipase C from Vibrio sp. and B. cereus. Itwill also be understood that, as an alternative to non-human lipases inpharmaceutical preparations from the current invention, lipases of humanorigin might be substituted or alternatively non-human lipases such asCPAT might be genetically engineered or modified in order to escaperecognition by the human immune system.

Where two or more lipase components are used in the method of thepresent invention or for substances and materials thereof, it might bepossible to improve the strength of association of these lipasecomponents in order to more quickly or more fully reconstitute enzymeactivity. This might be achieved by, for example, genetic engineering orby use of auxiliary components which themselves associate thus bringingthe lipase components together.

Thus CPAT or reconstituted CPAT activity may be used to direct the lysisof synthetic liposomes containing biologically active preparations ordetectable molecule such as dyes thereby providing inter alia amechanism for targeted drug delivery in vivo, or a reporter system forhomogenous and discontinuous assay systems in vitro.

A range of alternative liposomes may be applicable for use in thecurrent invention—the invention embraces all types of liposome.

The methods of the present invention will now be described, by way ofillustration only, through reference to the following examples andfigures. Other embodiments falling within the scope of the inventionwill occur to those skilled in the art in the light of these.

Figures

FIG. 1 Demonstrates the ability of purified recombinant CPAT(holoenzyme) to induce liposome lysis in an experiment carried out asdescribed in Example 1.

FIG. 2 Demonstrates the comparative activity against liposomes of i)purified recombinant CPAT (holoenzyme); ii) recombinant N-CPAT; iii)recombinant C-CPAT and iv) mixed recombinant N- and C-CPAT. Theexperiment was carried out as described in Example 2.

FIG. 3 Shows a schematic diagram illustrating the in vivo anti-cancermethod described in Example 3.

FIG. 3b Shows a schematic diagram illustrating the in vivo anti-cancermethod similar to that described in Example 3 wherein the holoenzyme isconjugated to the antibody.

FIG. 4 Shows a schematic diagram illustrating the in vitroantibody-mediated antigen detection assay described in Example 5.

FIG. 5 Shows a schematic diagram illustrating the in vitro nucleic acidhybridisation assay described in Example 6.

FIG. 6 Shows binding of antibody-lipase to an antigen on a solid phaseand subsequent liposomal lysis.

FIG. 7 Shows binding of antibody-lipase to a nucleotide sequence on asolid phase and subsequent liposomal lysis.

EXAMPLES Example 1 Liposome Lysis By Recombinant CPAT Holoenzyme

The ability of purified recombinant CPAT (holoenzyme) to induce liposomelysis was tested using liposomes containing carboxyfluorosceinsubstrate, lysis being assessed using a fluoremetric assay.Carboxyfluoroscein release was measured for 60 minutes using CPATconcentrations between 1 pM and 1 μM.

The results shown in FIG. 1, clearly show that CPAT can induce liposomelysis.

Sphingomyelin liposomes containing 5-(6)-carboxyfluorescein wereprepared using the method described by Senior and Gregoriadis (1983) inliposome technology (Gregoriadis ed.) vol. 111 pp263-82, CRC Press, BocaRaton, Fla. A thin film of a mixture of sphingomyelin andβ-oleoyl-γ-cholesterol (1:1 w/w, 35.5 mg total) in chloroform was driedunder nitrogen and carboxyfluorescein (20 mM) added. The mixture wasplaced in a bath sonicator at 40° C. and then left at 22° C. for 1 hourbefore sonication (10×1 min with 0.5 min between cycles, 40° C.;Heatsystems XL-200 sonicator with 19 mm probe) to generate smallunilamellar vesicles. The free carboxyfluorescein was separated from theliposomes using gel filtration chromatography (Sephadex G-25; PharmaciaPD10 column) with borate buffered saline (BBS; 0.2 M sodium metaborate,7.5 g/l NaCl, 1.8 g/i CaCl₂.2H₂O, pH adjusted to 7.5 with boric acid) asthe eluting buffer. Fractions containing liposomes were collected andstored at 4° C.

Liposomes were diluted in BBS and 25 μl of enzyme added to 2.5 mlvolumes of the diluted liposomes. The mixture was incubated at 37° C.and fluorescence measured using a Perkin-Elmer L5-5B spectrofluorimeterwith exitation at 485 nm (10 nm slit width) and emission measured at 520nm (5 nm slit width).

The additon of C. perfringens α-toxin (phospholipase C) to6-carboxyfluorescein encapsulated liposomes of sphingomyelin produced atime-dependent increase in 6-carboxyfluorescein fluorescence. The rateof increase in fluorescence was related to the concentration of enzymeover the range tested (10 pM to 10 nm final concentration see FIG. 1).In the abscence of Ca²⁺ there was a four-fold decrease in the activityof the a-toxin (100 pM) against sphingomyelin liposomes.

The effect of Cpa₁₋₂₄₉ (N-domain) and Cpa₂₄₇₋₃₇₀ (C-domain) oncarboxyfluorescein release was that the individual domains of theα-toxin (Cpa₁₋₂₄₉ and Cpa₂₄₇₋₃₇₀) were incubated individually andtogether with liposomes containing 6-carboxyfluorescein. The resultsindicate that neither Cpa₁₋₂₄₉ nor Cpa₂₄₇₋₃₇₀ alone were able to induce6-carboxyfluorescein fluorescence from sphingomyelin liposomes. However,when both Cpa₁₋₂₄₉ and Cpa₂₄₇₋₃₇₀ were incubated with sphingomyelinliposomes an increase in fluorescence was detected.

Example 2 Comparative Activity of CPAT Holoenzyme and N- and C-Fragments

The comparative activity against liposomes of purified recombinant CPAT(holoenzyme). separate recombinant N- and C-CPAT, and mixed recombinantN-and C-CPAT was measured using a fluoremetric assay. In each case theconcentration was 100 pM of each protein.

The results, shown in FIG. 2, clearly show enhanced liposome degradationby combined N- and C-CPAT compared to the sum of their separateactivities.

Example 3 Method of Delivering Chemotherapeutics in Vivo

An in vivo anti cancer method was provided as shown in FIG. 3. In thismethod the invention is used to target chemotherapeutics at cancercells. C-CPAT (the smaller CPAT fragment) is conjugated to an antibodyspecific to a tumour (A) and is administrated in vivo. The conjugatedantibody binds at the tumour site (B) and the complementary (N-CPAT)fragment is administrated thereby causing lipase activity to beconstituted at the target cell surface (C). Liposomes containing achemotherapeutic (adriamycin) are administered (D) eithercontemporaneously with or subsequent to the N-CPAT. The restored CPATactivity at the tumour mediates liposomal lysis thereby achieving highlocal concentrations of chemotherapeutic agent (E).

In an alternative method the liposomes are used to deliveranti-inflammatory drugs or biological response modifiers to antigenpresenting cells or damaged tissues containing reactive infiltratingdisease lymphocytes.

It will be appreciated that the general methodology of Example 3 haswide application in the field of therapeutic medicine.

Example 4 Method of Genetically Modifying Cells in Vivo

An in vivo method for modifying the genetic nature of cells was providedas follows. The method is similar to that described in Example 3 exceptthat the liposomes are used to deliver anti-proliferative syntheticoligonucleotides to proliferating cells (such as endothelial cells).

It will be appreciated that the methodology of Example 4 may be used inthe treatment of many genetically based disorders e.g. cardiorestinosis,and will be of value in numerous therapeutic or experimental strategieswhich are dependent on the delivery of new genetic information to cellsin diverse disease settings.

Example 5 An Homogeneous in Vitro Analyte Detection Assay

An antibody-mediated in vitro analyte detection assay was provided asshown in FIG. 4.

First an analyte is conjugated to N-CPAT and the analyte/N-CPATconjugate is mixed with an antibody raised against the analyte whichbinds thereto. This complex is mixed with C-CPAT and liposomescontaining a reporter molecule in a system suspected of containinganalyte. In the absence of analyte the constitution of CPAT activity issterically prevented by the antibody and the liposomes remain intact(A). In the presence of analyte the antibody is sequestered from theN-CPAT complex thus allowing CPAT activity to be constituted andliposomal lysis to occur thereby releasing the reporter molecules (B).

It will be appreciated that such a detection system will have wideapplication in diagnostic and research fields.

Example 6 An in Vitro Nucleic Acid Hybridisation Assay

An in vitro nucleic acid sequence detection assay was provided asemploying conjugated probes as shown in FIG. 5.

Nucleic acid molecules (probes) capable of hybridising to the sequenceto be detected such that they are adjacent each other are conjugated toN-CPAT and C-CPAT. The conjugates are added to a system suspected ofcontaining the sequence. In the presence of the sequence hybridisationof the probes occurs thereby bringing the N-CPAT and C-CPAT intoproximity and restoring CPAT activity. The restoration is signalled byliposomal lysis and the release of detectable molecules such as dyes.

It will be appreciated that such a detection system may be applied inboth an homogenous assay (no solid phase or separation step) anddiscontinuous assay (where the target sequence is bound to a solid phaseformat).

FIG. 7 illustrates an in vitro nucleic acid detection assay. Anucleotide sequence 1 is attached to a solid phase 2 either directly orindirectly. The probe lipase component 3 comprising the lipase 4, whichmay be the complete lipase or one component, is then added followed byif required, the second lipase component. The probe lipase component 3also comprises a short nucleotide sequence 5 which is complementary topart of the nucleotide sequence 1. Liposomes 6 are added to the systemand are lysed on contact with the probe lipase component 3, thusreleasing the contents of the liposomes.

The lipase comprising C.perfringens alpha toxin N-terminal domaincorresponds to that which possesses phospholipase activity—typicallythis may include residues from 1-260 which in turn may include some orall of the amino acids in this range and the C-terminal domain whichconfers liposome lysing properties, typically may include residues from240 to 370 which in turn may include some or all of the amino acids inthis range.

The following experimental methods illustrate the killing of cancercells using liposome-conjugated antibodies in conjunction withliposome-encapsulated drugs.

Anti-CEA antibodies are targetted with conjugated Clostridiumperfringens phospholipase C holoenzyme to HeLa cells in vitro. Followingbinding of antibody lipase conjugate to the cells, excess conjugate isremoved and drug liposomes are added. The antibody-conjugatephospholipase C breaks open the liposomes, releasing the cytotoxic drug,leading to an enhanced killing of the cancer cells. Appropriate controlsare included to identify background cell death due to non-specificendocytosis of drug-liposomes or any possible lipase activity actingdirectly on the cancer cell.

Conjugation of Phospholipase C to Anti-CEA Antibody

Anti-CEA antibody was obtained from the Scottish Antibody ProductionUnit (Carluke, Scotland). Recombinant Clostridium perfringensphospholipase C was produced as described in the literature. Theanti-CEA antibody was conjugated to the phospholipase C using Sulfo-MBS(Pierce and Warriner, Chester, England) according to the manufacturer'sinstructions. Unconjugated antibody or phospholipase C was removed bydialysis.

Enhanced Killing of HeLa Cells Using Anti-CEA-Phospholipase C Conjugatein Conjunction with Drug Liposomes

100 ml of HeLa cells in DMEM+10% foetal calf serum were plated into eachwell of a 96 well tissue culture plate and cells were allowed to grow tosemi-confluency. The growth media was removed and replaced with 100 mlof various concentration of antibody-enzyme conjugate diluted in DMEM+10% FCS. The conjugate was allowed to bind with the cells for 3 hoursat 37° C. After this incubation the unbound Antibody-enzyme conjugatewas removed by washing 3× with DEME +10% FCS. After washing, 100 ml ofvarious concentrations of DaunoXomes (Nexstar Pharmaceutical Ltd,Cambridge, England) diluted in DMEM +10% FCS was added to the wells andincubation allowed to continue at 37° C. for 16-24 hours. An estimate ofthe number of viable cells was obtained using a ‘Celltiter 96 cellproliferation assay’ (Promega, Southampton, England).

At 1:100 dilution of liposomes, viable cells were reduced by <50%without phospholipase C or conjugated antibody. The addition ofapproximately 10 μg/ml conjugate resulted in a reduction of viable cellcount >50% whilst the addition of phospholipase C had no effect on theliposome induced reduction in viable cells. If liposomes were addeddirectly with phospholipase C or conjugated antibody to cells, then noreduction in viable cell count was obtained indicating destruction ofthe liposomes in solution.

What is claim is:
 1. A method for releasing at least one pharmaceuticalagent at a site of a target comprising the steps: A binding an antibodyto said target wherein said antibody is conjugated I) to a lipasewherein the lipase is able to lyse liposome, or II) to at least a firstlipase component having no or less lipase activity in comparison to acorresponding lipase holoenzyme, and B) I) wherein when said antibody isconjugated to a lipase able to lyse liposomes, contacting a liposomecontaining said pharmaceutical agent with said antibody conjugated to alipase such that the liposome can be lysed by the lipase, or II) whereinwhen said antibody is conjugated to the at least a first lipasecomponent, a) adding a second lipase component which reconstitutes thelipase activity of said at least a first lipase component at leastpartially by binding to the first lipase component and, subsequently,contacting a liposome containing said pharmaceutical agent with thereconstituted lipase such that the liposome can be lysed by thereconstituted lipase activity, or b) adding a liposome containing saidpharmaceutical agent to said antibody conjugated to the at least a firstlipase component wherein the liposome is associated with a second lipasecomponent able to reconstitute the lipase activity of the at least afirst lipase component by binding to the first lipase component whereinsaid second lipase component is an integral part of the liposome.
 2. Amethod as claimed in claim 1 wherein unbound antibody which isconjugated to the first lipase component is cleared prior to adding thesecond lipase component or the liposomes associated with the secondlipase component.
 3. A method as claimed in claim 1 wherein the targetsite is a tumor, a diseased cell or a pathogenic organism.
 4. A methodas claimed in claim 1 wherein the pharmaceutical agent is selected fromthe group consisting of a reporter molecule, a chemotherapeuticcytotoxic drug, a lymphokine, an anti-inflammatory, an anti-fungalagent, or an anti-malarial agent.
 5. A method of delivering achemotherapeutic comprising a method as claimed in claim 1 wherein theagent is a chemotherapeutic cytotoxic drug.
 6. A method as claimed inclaim 5 for killing of neoplastic cells and/or inhibiting growth ofneoplastic cells.
 7. A method of delivering an anti-inflammatory drugcomprising a method as claimed in claim 1 wherein the agent is ananti-inflammatory drug.
 8. A method as claimed in claim 1 wherein thelipase activity comprises phospholipase C activity.
 9. A method asclaimed in claim 8 wherein the phospholipase C activity comprises CPAT.10. A method as claimed in claim 1 wherein the lipase components areN-terminal recombinant CPAT and C-terminal recombinant CPAT.